Imperial Centre for Translational and Experimental Medicine, Department of Surgery & Cancer, Imperial College London, London, UK.
Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, Sutton, UK.
Mol Cancer. 2022 Mar 22;21(1):82. doi: 10.1186/s12943-022-01540-w.
miR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent.
RNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq).
miR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival. INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes. NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy.
A balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.
miR-346 被鉴定为雄激素受体 (AR) 信号的激活剂，与前列腺癌 (PC) 中与 DNA 损伤反应 (DDR) 相关的转录本相关联。我们试图描绘 miR-346 对 DNA 损伤的影响及其作为治疗剂的潜力。
使用 RNA-IP、RNA-seq、RNA-ISH、DNA 纤维分析、体内异种移植研究和生物信息学方法，以及一种用于扩增自由、单核苷酸分辨率全基因组 DNA 断裂图谱的新方法（INDUCE-seq）。
miR-346 在 PC 细胞中诱导快速和广泛的 DNA 损伤-这是 miRNA 诱导的 DNA 损伤的首次报道。从机制上讲，这是通过转录超激活、R 环形成和复制应激来实现的，导致检查点激活和细胞周期停滞。miR-346 还与基因组保护 lncRNA NORAD 相互作用，破坏其与 PUM2 的相互作用，导致 PUM2 稳定及其对 DNA 损伤反应 (DDR) 转录本的增加。证实临床相关性，NORAD 表达和活性与 PC 临床结果不良和活检 RNA-seq 研究中 DDR 增加强烈相关。相比之下，miR-346 与改善的 PC 生存相关。INDUCE-seq 显示，miR-346 诱导的 DSBs 优先发生在 PC 细胞中最具转录活性的转录因子的结合位点上，包括 c-Myc、FOXA1、HOXB13、NKX3.1，并且重要的是 AR，导致靶转录物下调。此外，RNA-seq 显示 miR-346 和 shNORAD 广泛失调 DNA 损伤、复制和细胞周期过程。NORAD 驱动 miR-346 的靶向导向 miR 衰减 (TDMD) 作为一种新的基因组保护机制：NORAD 沉默使成熟 miR-346 水平增加几千倍，并且 WT 但不是 TDMD 突变 NORAD 可挽救 miR-346 诱导的 DNA 损伤。重要的是，miR-346 使 PC 细胞对包括 PARP 抑制剂和化疗在内的 DNA 损伤药物敏感，并在体内作为单一疗法诱导肿瘤消退，表明靶向 miR-346：NORAD 平衡是一种有效的治疗策略。
miR-346 和 NORAD 之间的平衡作用调节 PC 中的 DNA 损伤和修复。miR-346 在观察到的晚期 PC 中 NORAD 减少的情况下，以及在转录活性高的癌细胞中，可能特别有效作为治疗剂。
